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Total RNA Isolated (total + rna_isolated)
Selected AbstractsExpression of metalloproteinases and their tissue inhibitors in inflamed gingival biopsiesJOURNAL OF PERIODONTAL RESEARCH, Issue 5 2008L. D. R. Gonçalves Objectives:, Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are known to be involved in the periodontal disease process. Results of in vivo MMPs and TIMPs gene expressions in the gingiva, though, are still controversial. In the present study, we compared the gene expression of MMP-1, -2, -9, -13 and TIMP-1, -2 in healthy and inflamed gingiva. Methods:, 38 gingival samples were collected from gingivitis (n = 10), advanced chronic periodontitis (n = 10), generalized aggressive periodontitis (n = 8) and periodontally healthy individuals (n = 10). Total RNA isolated from those samples was subjected to reverse transcription followed by amplification by polymerase chain reaction (RT-PCR). Products were visualized in agarose gels and quantified by optical densitometry. Samples were also processed for gelatin zymography and Western blotting for MMP-2 and MMP-9 in order to assess for post-transcriptional MMP regulation at the protein level. Results:, The frequencies and levels of transcripts encoding MMPs and TIMPs were found to be not significantly different among groups (p > 0.05, Fisher's Exact and Kruskall-Wallis tests). There is a trend towards higher MMP-2 and -9 gelatinase activities in the inflamed samples, although not statistically significant. In contrast, zymography and Western blotting studies show that MMP-2 is virtually absent in the chronic periodontitis group. Conclusion:, These results could reflect a complex regulation of MMPs and TIMPs' gene expression in the course of gingival inflammation. They also reveal a great biological diversity even among individuals with similar periodontal status. [source] Progressive up-regulation of genes encoding DNA methyltransferases in the colorectal adenoma-carcinoma sequenceMOLECULAR CARCINOGENESIS, Issue 9 2007Wolfgang M. Schmidt Abstract Epigenetic silencing is a prominent feature of cancer. Here, we investigated the expression of DNA demethylase and three DNA methyltransferases during colorectal tumorigenesis comparing the genes encoding DNA methyltransferases 1 (DNMT1), 3A, and 3B (DNMT3A and DNMT3B) with methyl-CpG binding domain protein 2 (MBD2), recently described as the only active DNA demethylase. Total RNA isolated from normal colonic mucosa (n,=,24), benign adenomas (n,=,18), and malignant colorectal carcinomas (n,=,32) was analyzed by reverse transcriptase-PCR with subsequent quantification by capillary gel electrophoresis. In contrast to MBD2, expression of DNMT1 and DNMT3A increased in parallel to the degree of dysplasia, with significant overexpression in the malignant lesion when compared with mucosa or with benign lesions (DNMT1). Pairwise comparisons between tumors and matched, adjacent healthy mucosa tissue (n,=,13) revealed that expression of all three genes encoding DNA methyltransferases increased by two- to three-fold. Our data suggest a relevant role of the DNA methyltransferases during colorectal tumorigenesis. This increase is not counterbalanced by enhanced expression of the demethylating component MBD2. As a consequence, epigenetic regulation in the adenoma-carcinoma sequence may be driven by increased methylating activity rather than suppressed demethylation. © 2007 Wiley-Liss, Inc. [source] Evaluation of malignant and benign gastric biopsy specimens by mRNA expression profile and multivariate statistical methodsCYTOMETRY, Issue 5 2007Orsolya Galamb Abstract Background: mRNA expression array and multivariate statistical analysis of gastric biopsies can yield insight into the molecular biology basis of local alterations, supporting expression-based identification of morphological alterations. Methods: From 11 patients with erosive gastritis(EG), 5 with adenocarcinoma (GC), 11 with atrophic gastritis (AG) gastric biopsies were collected, total RNA isolated, T7 amplification and expression analysis of 1047 mRNAs was performed using commercial glass arrays (Clontech, USA). After microarray quality control, applicable data were available from 7 EG, 4 GC, and 5 AG. Multivariate statistical and cell functional analysis were performed. Real-time RT-PCR and immunohistochemistry were used for validation. Results: GC was characterized by overregulated v-raf, v-erb-a, BCL2-associated- athanogene, immediate-early-response-3, Polo-like kinase, CDK-2, cyclin-C, Pin1 genes, and downregulated ADP-ribosyltransferase, sialophorin and DCC. AG cases had increased PDGF-receptor, TGF-,-receptor-3, and decreased death-associated-protein-3, ,-1-catenin, topoisomerase-1 levels. In EG upregulation of IGF-receptor-1, CD9, transferrin receptor, integrins, and underexpression of keratin-5, caspase-4 was found. Discriminant analysis could reclassify all samples correctly using four parameters. Conclusions: mRNA expression array analysis of gastric biopsies yields previously known and new data in the evaluation of local gastric alterations. © 2007 Clinical Cytometry Society [source] Reactive oxygen species induce RNA damage in human atherosclerosisEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 5 2004W. Martinet Abstract Background, Reactive oxygen species (ROS)-induced DNA damage has recently been identified in both human and experimental atherosclerosis. This study was undertaken to investigate whether RNA damage occurs in human atherosclerotic plaques and whether this could be related to oxidative stress. Materials and methods, The integrity of total RNA isolated from carotid endarterectomy specimens (n = 20) and nonatherosclerotic mammary arteries (n = 20) was analyzed using an Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA). Oxidative modifications of RNA were detected by immunohistochemistry. Results, Eleven out of 20 atherosclerotic plaques showed a significant reduction of the 18S/28S rRNA peaks and a shift in the RNA electropherogram to shorter fragment sizes. In contrast, all mammary arteries showed good-quality RNA with clear 18S and 28S rRNA peaks. Strong nuclear and cytoplasmic immunoreactivity for oxidative damage marker 7,8-dihydro-8-oxo-2,-guanosine (8-oxoG) could be detected in the entire plaque in smooth muscle cells (SMCs), macrophages and endothelial cells, but not in SMCs of adjacent normal media or in mammary arteries. Cytoplasmic 8-oxoG staining in the plaque clearly diminished when tissue sections were pretreated with RNase A, suggesting oxidative base damage of RNA. In vitro treatment of total RNA with ROS-releasing compounds induced RNA degradation. Conclusion, Both loss of RNA integrity and 8-oxoG oxidative modifications were found in human atherosclerotic plaques. Because RNA damage may affect in vitro transcript quantification, RT-PCR results must be interpreted cautiously if independent experimental validation (e.g. evaluation of RNA integrity) is lacking. [source] Characterization of t(6;11)(p21;q12) in a renal-cell carcinoma of an adult patientGENES, CHROMOSOMES AND CANCER, Issue 5 2007Lorenza Pecciarini Renal-cell carcinoma (RCC) constitutes a heterogeneous group of tumors with specific chromosome aberrations. Recently, a new small group of RCC, occurring in children and young adults, has been described as characterized by t(6;11)(p21;q12). It has been shown that this translocation results in the fusion of the 5, portion of the ALPHA gene (11q12) with the transcription factor gene TFEB (6p21). Herewith, we report the first complete cytogenetic and molecular characterization of a t(6;11)-positive RCC of an adult patient, a 54-year-old woman. The tumor was histologically defined as RCC with peculiar features and it was negative for epithelial markers and positive for melanocytic markers. Chromosome QFQ banding analysis of short-term cultured cells from the RCC showed t(6;11)(p21;q12) as the sole cytogenetic abnormality. The translocation was confirmed by FISH analysis. RT-PCR analysis, performed on total RNA isolated from both neoplastic and normal tissue samples, revealed an ALPHA,TFEB chimeric transcript in the tumor sample; sequencing of the RT-PCR product defined a novel TFEB gene breakpoint cluster region, broader than the one reported thus far. Western blot analysis showed a band at the expected size of wild-type TFEB in the neoplastic tissue compared to the normal sample, supporting that the fusion gene does not encode for a chimeric protein but it causes an upregulation of the wild-type TFEB. Our data contribute to define better this rare RCC type, which is typical not only of childhood but can also be found in adulthood. © 2007 Wiley-Liss, Inc. [source] Identification and quantification of differentially expressed transcripts in in vitro-produced bovine preimplantation stage embryosMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2003Dawit Tesfaye Abstract In this study, we used mRNA differential display reverse transcription polymerase chain reaction (DDRT-PCR) to analyze the mRNA expression patterns in in vitro-produced bovine 8-cell, 16-cell, morula, and blastocyst stage embryos and isolate differentially expressed amplicons. Moreover, we have used a fluorescence monitored real time quantitative PCR to quantify and analyze the expression patterns of the target differentially expressed transcripts through out the preimplantation stages from oocytes to blastocyst. For this, total RNA isolated from bovine 8-cell (n,=,188), 16-cell (n,=,94), morula (n,=,35), and blastocyst (n,=,15) were reverse transcribed and subjected to DDRT-PCR. Target differentially expressed transcripts were quantified by real time quantitative PCR. The cDNA banding pattern analysis revealed that large number of cDNA bands were conserved at 8-cell and blastocyst stage with a slight decrease at the morula stage. A total of 16 amplicons were cloned and sequenced. All expressed sequence tags (ESTs), except 1C19, showed sequence similarity with known genes or ESTs in GenBank. Sixty-two percent (10/16) of cDNA bands representing differentially expressed genes originated from 8-cell stage and the rest derived from the 16-cell, morula, or blastocyst stage. The quantitative PCR analysis has validated the expression patterns of 75% (12/16) of our transcripts to be in agreement with the results of DDRT-PCR. However, the quantitative PCR results of four transcripts showed a deviation from the pattern seen in DDRT-PCR. In conclusion, we have successfully applied mRNA DDRT-PCR to identify and isolate stage-specific expressed genes in bovine preimplantation embryos. In addition to validating the results of DDRT-PCR, quantitative real time PCR provides quantitative data on the expression of target genes. Mol. Reprod. Dev. 66: 105,114, 2003. © 2003 Wiley-Liss, Inc. [source] | |||||||||||||